The active site of a number of metalloproteins and enzymes contains a coupled binuclear copper unit which exhibits similar chemical and spectroscopic properties and interacts with oxygen as part of its biological function. This coupled binuclear copper active site is distributed in a wide variety of metalloproteins which perform vital roles in copper and iron metabolism, oxygen activation, and electron transfer. In different metalloproteins, this site cooperatively binds oxygen (arthropod and mollusc heymocyanin), dismutates peroxide (mollusc hemocyanin), hydroxylates monophenols (tyrosinase) and, in conjunction with several other copper centers, couples four one-electron oxidations of substrate to the four-electron reduction of dioxygen to water (laccase, ceruloplasmin and ascorbic acid oxidase). The objective of this proposed research is to study in detail the chemistry and spectroscopy of this coupled binuclear copper site with the purpose of understanding its unique spectral features and thus its electronic and geometric structure and the correlation of these features with protein function. Our approach has involved the development of a series of protein derivatives which enable the coupled binuclear copper active site to be systematically varied and subjected to detailed spectroscopic study. The techniques which are emphasized include X-and Q-band EPR, absorption (with linearly polarized single crystal methods), CD, variable excitation energy resonance Raman and variable temperature MCD spectroscopy. Very specific experiments also involve electron spin echo and ENDOR, x-ray absorption and EXAFS, Fourier Transform IR and NMR and magnetochemistry with a SQUID system. Our work thus far has enabled us to interpret the unique spectral features of oxyhemocyanin and generate a "spectroscopically effective" picture of this active site. Further, our chemical and spectroscopic studies have provided preliminary insight into the strong similarities between the coupled binuclear copper active site in hemocyanin and tyrosinase and the significant differences present in the binuclear type 3 copper site in laccase. Detailed comparative studies of this coupled binuclear copper active site in the metalloproteins and enzymes listed above should now allow a correlation of differences in this structure with variation in protein function.